Document Type and Release Option

Copyright Statement / License for Reuse

Department

Department of Biology

Committee Chair

Oscar Pung

Committee Member 1

Lance Durden

Committee Member 2

Quentin Fang

Abstract

Trematodes infect over 2 billion people and are responsible for annual livestock losses of at least $2 billion. In vitro cultivation of adult trematodes is a good way for scientists to study the biology of these parasites and develop new drugs and vaccines. Successful in vitro growth of excysted metacercariae into mature egg-producing adults requires that the worms be fertilized. The goals of the present study were to: (1) determine the length of time needed for in vitro sperm development in excysted metacercariae of the trematode Gynaecotyla adunca, (2) optimize an in vitro fertilization protocol for the parasite, and (3) test the effect of this protocol on the production of eggs in culture. Microphallus turgidus has been successfully cultured in vitro (Pung, et al., 2011) and was used in this study as a control. Metacercarial cysts of G. adunca, were obtained from the green glands of fiddler crabs, Uca pugnax, excysted in 0.5% trypsin, washed and incubated in HBSS. Excysted worms were examined microscopically to determine sperm maturation time. The effect of different culture vessels and worm density on fertilization was tested. Temperature, incubation time, and pH were also examined. Worms fertilized in the optimal combination of the above conditions were cultured in DME/F-12 with horse serum for 7 days at 42 C and the number of eggs deposited was counted. Sperm maturation time for G. adunca was 8 – 10 hr post excystment. The highest percentage of worms fertilized in culture was observed when 50 excysted metacercariae were incubated in 15 ml conical bottom tubes at 37 C, pH 7 for 48 hr. Worms fertilized in these conditions deposited higher numbers of normal-shaped, embryonated eggs when subsequently cultured.